László Csanády

The Ca ²⁺ permeable cation channel TRPM2 is expressed in neurons of the hypothalamic thermoregulatory center, and contributes to body temperature regulation. Its activation gate opens upon binding of cytosolic Ca ²⁺ and ADP ribose, but its distinct inactivation gate closes upon prolonged ligand exposure. In contrast to inactivation of most other neuronal channels, inactivation of human TRPM2 (hsTRPM2) is irreversible, but neither the mechanistic reason nor the evolution of that unique feature is known. Here we find that for the zebrafish ortholog (drTRPM2) inactivation is readily reversible upon ligand removal. Using a combination of electrophysiology, kinetic modeling, and thermodynamic analysis we reveal strong allosteric coupling between the extracellular inactivation gate and the cytosolic Ca ²⁺ binding site: inactivation gate closure traps Ca ²⁺ in its binding site. Moreover, we demonstrate that for hsTRPM2 inactivation is also technically reversible, but hsTRPM2 channels have evolved to bind Ca ²⁺ so tightly in the inactivated state, that recovery requires lowering free [Ca ²⁺ ] to subnanomolar levels. These findings delineate the appearance and evolution of TRPM2 inactivation in vertebrates, and explain why the human channel remains trapped in the inactivated state under physiological conditions.

The epithelial anion channel cystic fibrosis transmembrane conductance regulator (CFTR) is activated by cAMP‐dependent protein kinase (PKA). PKA stimulates CFTR channels through two mechanisms: non‐catalytically, by binding to the channel, and catalytically, by phosphorylating its regulatory (R) domain. CFTR mutations that reduce channel activity cause cystic fibrosis (CF), but clinically used modulator drugs that boost channel function can alleviate disease symptoms. The two common CF mutations, ΔF508 and G551D, have been reported to impair CFTR channel activation by PKA, but the mechanisms remain unclear. Here, we aimed to understand how the mutations impact non‐catalytic vs . catalytic channel activation by PKA and how these two processes are modulated by clinically used potentiator drugs. Using current recordings from excised inside‐out membrane patches superfused with the purified catalytic subunit of PKA, we confirm slowed PKA‐dependent activation for both mutants but demonstrate intact binding affinity for the kinase. Furthermore, we find that non‐catalytic activation dominates overall channel activity for both mutants and can be strongly enhanced by stabilization of the NBD1–NBD2–TMD interface using the ATP analogue N ⁶ ‐(2‐phenylethyl)‐ATP. For both mutants, the clinically used potentiator drug combination elexacaftor + ivacaftor boosts catalytic channel activation by PKA more efficiently than non‐catalytic activation. For ΔF508 CFTR, elexacaftor + ivacaftor evokes substantial PKA‐independent channel activity and entirely suppresses non‐catalytic activation by PKA. These findings help us to understand the activation defects caused by two common CF mutations and suggest room for further improvement of potentiator drugs currently used in CF therapy. image Key points: Protein kinase A (PKA) activates the epithelial anion channel cystic fibrosis transmembrane conductance regulator (CFTR) through two mechanisms: non‐catalytically, by binding to the channel, and catalytically, by phosphorylating its regulatory (R) domain. CFTR mutations cause cystic fibrosis (CF); the two common mutations, ΔF508 and G551D, reportedly also impair channel activation by PKA. We show here, for both mutants, that PKA‐dependent activation is slowed despite intact binding affinity for the kinase, and that non‐catalytic activation dominates overall channel activity and might be further enhanced by stabilization of the NBD1–NBD2–TMD interface. For ΔF508 CFTR, but not for G551D CFTR, a combination of clinically used potentiator drugs evokes substantial PKA‐independent channel activity but suppresses non‐catalytic activation by PKA. These findings help us to understand the activation defects caused by two common CF mutations and suggest room for further improvement of potentiator drugs currently used in CF therapy.

TRPM2 is a Ca ²⁺ permeable cation channel activated by cytosolic Ca ²⁺ and ADP ribose (ADPR). In contrast to invertebrate orthologs, vertebrate TRPM2 channels inactivate due to evolutionary alterations in amino acid sequence around the selectivity filter. Human TRPM2 (hsTRPM2) serves as a deep-brain temperature sensor important for body temperature regulation, and its gating is exquisitely temperature dependent. To address whether TRPM2 is temperature sensitive also in ectotherms that lack body temperature regulation, here we investigated the functional properties of zebrafish ( Danio rerio ) TRPM2 (drTRPM2) across the temperature range of 15°C to 37°C. The rate of ion permeation through the open pore was weakly temperature sensitive (Q ₁₀ ∼1.3) as expected for a diffusion-limited process. In the presence of saturating concentrations of ligands (ADPR and Ca ²⁺ ) drTRPM2 open probability showed no temperature dependence. Moreover, for both ADPR and Ca ²⁺ , the apparent affinities for channel activation were unaffected by temperature, reporting a standard enthalpy of opening near zero for drTRPM2. Inactivation of drTRPM2 is an order of magnitude slower than that of hsTRPM2. To address whether in both orthologs the same mechanism underlies inactivation, we studied its temperature sensitivity. For both drTRPM2 and hsTRPM2 inactivation rate was modestly temperature dependent (Q ₁₀ ∼2.7 and ∼4.4). A triple substitution which converts the post-filter sequence of drTRPM2 into the corresponding human sequence accelerated drTRPM2 inactivation by >20-fold. The data suggest that temperature dependence of hsTRPM2 gating evolved in the course of vertebrate evolution, whereas inactivation was temperature dependent already when it first appeared in early vertebrates.

Cystic fibrosis (CF) is a life‐threatening condition caused by pathogenic mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel. While most patients with frequent mutations are treated with a combination of corrector and potentiator drugs (elexacaftor–tezacaftor–ivacaftor, ETI), for many rare variants no such treatment is approved. Here we characterized the molecular pathologies of five rare CF‐associated mutants (G126D, I336K, T465I, T582I and D984V), and evaluated their responsiveness to ETI treatment. Channel expression was investigated by western blot from HEK‐293T cells transiently expressing mutant channels, with or without corrector drugs (elexacaftor and tezacaftor). Drug efficiency was assessed by the enhancement of channel glycosylation. Maturation was mildly (T582I, G126D) to severely (T465I) defective for all five mutants, but could be restored to wild‐type levels by incubation with corrector drugs. Gating properties and responses to potentiator drugs (ivacaftor and elexacaftor) were assessed in macroscopic and single‐channel inside‐out patch‐clamp recordings from mutant channels expressed in Xenopus laevis oocytes. Mutations G126D, T582I, I336K and T465I markedly decreased channel open probability, with the greatest reduction being caused by mutation T465I. Mutations G126D, I336K and T465I also slightly impaired unitary conductance. Applying potentiator drugs boosted gating for all variants, producing multifold increases in macroscopic currents. In conclusion, all five mutations impair channel maturation and gating to various degrees, but a normal glycosylation pattern can be restored with correctors, and channel gating can be enhanced with potentiators. These in vitro observations suggest that ETI treatment would be beneficial for CF patients carrying an allele with any of the five mutations. image Key points: Mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel cause cystic fibrosis. Highly efficient modulator therapy has been approved for ∼90% of cystic fibrosis patients, but a large number of rare mutations are not yet eligible for treatment. Five rare CFTR mutations found in cystic fibrosis patients in Hungary and worldwide are shown here to produce complex channel pathologies that to differing degrees affect protein maturation, channel gating and anion permeation through the open pore. All five variants respond strongly to clinically employed modulator drugs that boost channel surface expression and gating. The results help us understand how these CFTR mutations lead to cystic fibrosis, and suggest that patients carrying any of the five variants would probably benefit from modulator therapy.

Transient Receptor Potential Melastatin 2 (TRPM2) cation channels contribute to immunocyte activation, insulin secretion, and central thermoregulation. TRPM2 opens upon binding cytosolic Ca ²⁺ and ADP ribose (ADPR). We present here the 2.5 Å cryo-electronmicroscopy structure of TRPM2 from Nematostella vectensis (nvTRPM2) in a lipid nanodisc, complexed with Ca ²⁺ and ADPR-2′-phosphate. Comparison with nvTRPM2 without nucleotide reveals that nucleotide binding-induced movements in the protein’s three “core” layers deconvolve into a set of rigid-body rotations conserved from cnidarians to man. By covalently crosslinking engineered cysteine pairs we systematically trap the cytosolic layers in specific conformations and study effects on gate opening/closure. The data show that nucleotide binding in Layer 3 disrupts inhibitory intersubunit interactions, allowing rotation of Layer 2 which in turn expands the gate located in Layer 1. Channels trapped in that “activated” state are no longer nucleotide dependent, but are opened by binding of Ca ²⁺ alone.

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), the anion channel mutated in cystic fibrosis (CF) patients, is activated by the catalytic subunit of protein kinase A (PKA-C). PKA-C activates CFTR both noncatalytically, through binding, and catalytically, through phosphorylation of multiple serines in CFTR’s regulatory (R) domain. Here, we identify key molecular determinants of the CFTR/PKA-C interaction essential for these processes. By comparing CFTR current activation in the presence of ATP or an ATP analog unsuitable for phosphotransfer, as well as pseudosubstrate peptides of various lengths, we identify two distinct specific regions of the PKA-C surface which interact with CFTR to cause noncatalytic and catalytic CFTR stimulation, respectively. Whereas the “substrate site” mediates CFTR phosphorylation, a distinct hydrophobic patch (the “docking site”) is responsible for noncatalytic CFTR activation, achieved by stabilizing the R domain in a “released” conformation permissive to channel gating. Furthermore, by comparing PKA-C variants with different posttranslational modification patterns, we find that direct membrane tethering of the kinase through its N-terminal myristoyl group is an unappreciated fundamental requirement for CFTR activation: PKA-C demyristoylation abolishes noncatalytic, and profoundly slows catalytic, CFTR stimulation. For the F508del CFTR mutant, present in ~90% of CF patients, maximal activation by demyristoylated PKA-C is reduced by ~10-fold compared to that by myristoylated PKA-C. Finally, in bacterial genera that contain common CF pathogens, we identify virulence factors that demyristoylate PKA-C in vitro, raising the possibility that during recurrent bacterial infections in CF patients, PKA-C demyristoylation may contribute to the exacerbation of lung disease.

Protein kinase A (PKA) is a key regulator of cellular functions by selectively phosphorylating numerous substrates, including ion channels, enzymes, and transcription factors. It has long served as a model system for understanding the eukaryotic kinases. Using cryoelectron microscopy, we present complex structures of the PKA catalytic subunit (PKA-C) bound to a full-length protein substrate, the cystic fibrosis transmembrane conductance regulator (CFTR)—an ion channel vital to human health. CFTR gating requires phosphorylation of its regulatory (R) domain. Unphosphorylated CFTR engages PKA-C at two locations, establishing two “catalytic stations” near to, but not directly involving, the R domain. This configuration, coupled with the conformational flexibility of the R domain, permits transient interactions of the eleven spatially separated phosphorylation sites. Furthermore, we determined two structures of the open-pore CFTR stabilized by PKA-C, providing a molecular basis to understand how PKA-C stimulates CFTR currents even in the absence of phosphorylation.

CFTR, the anion channel mutated in cystic fibrosis (CF) patients, is activated by the catalytic subunit of protein kinase A (PKA-C). PKA-C activates CFTR both reversibly, through binding, and irreversibly, through phosphorylation of multiple serines in CFTR’s regulatory (R) domain. Here we identify key molecular determinants of the CFTR/PKA-C interaction essential for these processes. By comparing CFTR current activation in the presence of ATP or an ATP analog unsuitable for phosphotransfer, as well as pseudosubstrate peptides of various lengths, we identify two distinct specific regions of the PKA-C surface which interact with CFTR to cause reversible and irreversible CFTR stimulation, respectively. Whereas the “substrate site” mediates CFTR phosphorylation, a distinct hydrophobic patch (the “docking site”) is responsible for reversible CFTR activation, achieved by stabilizing the R domain in a “released” conformation permissive to channel gating. Furthermore, by comparing PKA-C variants with different posttranslational modification patterns we find that direct membrane tethering of the kinase through its N-terminal myristoyl group is an unappreciated fundamental requirement for CFTR activation: PKA-C demyristoylation abolishes reversible, and profoundly slows irreversible, CFTR stimulation. For the F508del CFTR mutant, present in ∼90% of CF patients, maximal activation by de-myristoylated PKA-C is reduced by ∼10-fold compared to that by myristoylated PKA-C. Finally, in bacterial genera that contain common CF pathogens we identify virulence factors that demyristoylate PKA-C in vitro , raising the possibility that during recurrent bacterial infections in CF patients PKA-C demyristoylation may contribute to the exacerbation of lung disease. Significance Statement: CFTR is an anion channel crucial for salt-water transport across epithelia, and is activated by the catalytic subunit of protein kinase A (PKA-C). Reduced activity of mutant CFTR causes cystic fibrosis and CFTR hyperstimulation by sustained PKA-C activity causes diarrhea. PKA-C activates CFTR reversibly through simple binding, and irreversibly by phosphorylating the channel. We uncover here important structural requirements for these two processes. First, two distinct PKA-C surface areas mediate reversible and irreversible CFTR activation. Second, membrane anchoring of PKA-C through a covalently linked fatty (myristic) acid is required for both effects. Finally, we identify bacterial enzymes that cleave the myristic acid from PKA-C, thereby reducing activation of mutant CFTR channels, present in cystic fibrosis patients, by up to tenfold.